Environmental stress conditions, such as shortage or excess of solar energy, water or nutrients, high salinity and pollution (e.g., heavy-metal pollution), can have a major impact on plant growth and can significantly reduce plant yield. Osmotic stress, a type of environmental stress, may be induced by conditions of excess salinity, drought, excessive heat, cold or freezing.
Cold stress may be induced by temperatures below the range which allow optimal growth for a particular plant species. Each plant species or variety has an optimal growth temperature at which the growth rate is maximal; the further the deviation from this optimal growth temperature, the greater the stress on the plants. Many plant species, especially from tropical or subtropical regions, are sensitive to cold. For example, it has been estimated that the worldwide rice production would decrease by 40% if the worldwide mean temperature dropped only between 0.5 to 1.0° C. (Salisbury and Ross, Plant Physiology. 4th ed. Wadsworth Publishing Company, Belmont, Calif., 1992). Plants from temperate regions however have the ability to adapt their metabolism and to survive freezing temperatures after undergoing a process of adaptation to low but non-freezing temperatures, a process called cold acclimation. For instance non-acclimated rye typically does not survive temperatures below −5° C., but after cold acclimation it can withstand temperatures as low as −30° C. The process of cold acclimation involves altered expression of many genes. Plants may differ in their ability to withstand cold, which could lead to periodic but significant losses in plant productivity. As a consequence, the areas in which crops or horticultural plants can be cultivated is determined by assessing the risk of lower temperatures, relative to typical growth temperatures for any given plant.
The most prominent changes during cold acclimation include a reduction or cessation of growth, reduction of tissue water content (Levitt; Responses of Plants to Environmental Stresses, Vol. 1. 2nd edn. Academic Press. New York, N.Y. 1980), transient increase in abscisic acid (ABA) levels (Chen et al., Plant Physiology 71, 362-365, 1983), changes in membrane lipid composition (Lynch and Steponkus, Plant Physiology 83, 761-767, 1987; Uemura and Steponkus, Plant Physiology 104, 479-496, 1994), the accumulation of compatible osmolytes such as proline, betaine, polyols and soluble sugars, and increased levels of antioxidants (Kostero and Lynch, Plant Physiology 98, 108-113, 1992; Kishitani et al., Plant, Cell and Environment 17, 89-95, 1994; Murelli et al., Physiologia Plantarum 94, 87-93 1995; Nomura et al., Euphytica 83, 247-250, 1995; Dörffling et al., Plant Molecular Biology 23, 221-225, 1997; Tao et al., Cryobiology 37, 38-45, 1998).
Various methods for the identification and isolation of genes or proteins differentially expressed during cold stress are known. For example, mapping techniques allow determination of chromosome locations of genes involved in cold tolerance (Pan et al., Theoretical and Applied Genetics 89, 900-910, 1994; Galiba et al., Theoretical and Applied Genetics 90, 1174-1179, 1995). Another approach involves mutational analysis in which mutants that have an altered response to cold tolerance are isolated and characterized. For example, eskimol, conferring improved freezing tolerance of 2° C. over acclimated wild-type plants, was isolated from a collection of 800000 Ethyl Methyl Sulphonate (EMS)-mutagenised Arabidopsis lines that were screened for constitutively freezing-tolerant mutants (Xin and Browse, PNAS 95, 7799-7804, 1998). Conversely, plant lines were screened for mutants defective in cold acclimation (Warren et al., Plant Physiology 111, 1011-1019, 1996; Knight et al., Plant Cell 8, 489-503, 1996). cos-, los- and hos-mutants (for respectively constitutive, low and high expression of osmotically responsive genes) were isolated using a combination of mutagenesis and reporter gene activation (Ishitani et al., Plant Cell 9, 1935-1949, 1997; Ishitani et al., Plant Cell 10, 1151-1161, 1998; Lee et al., Plant Journal 17, 301-308, 1999). One of the drawbacks of mapping and the mutant analysis strategy is that they do not directly result in the isolation of nucleic acids coding for cold-induced genes. Another strategy, using differential screening of cDNA libraries and related techniques, has in the past yielded several cold induced genes from different plant species (reviewed in Xin and Browse, Plant, Cell and environment 23, 893-902, 2000). Many of those genes have known functions and can be grouped as being involved in drought stress, in signal transduction pathways, or as being related to heat shock proteins, molecular chaperones, “antifreeze proteins” or regulatory proteins. Several of the genes are highly expressed during cold stress and are commonly referred to as COR (COld Regulated) genes (Tomashow, Annual Review of Plant Physiology and Plant Molecular Biology 50, 571-599, 1999).
Strategies used to engineer cold resistant plants include accumulation of osmoprotectants such as mannitol (U.S. Pat. No. 6,416,985), proline (U.S. Pat. No. 6,239,332), trehalose (U.S. Pat. No. 6,323,001) or glycine-betaine (Hayashi et al., Plant Journal 12, 133-142, 1997; U.S. Pat. No. 6,281,411). Other approaches involve manipulating the signal transduction pathway controlling the stress response (WO 01/77355), including use of transcription factors (WO 01/77311, U.S. Pat. No. 6,417,428, WO 02/44389, U.S. Pat. No. 5,891,859). Furthermore a number of genes have been used to enhance cold resistance. Examples are members of the COR group (COR15a: U.S. Pat. Nos. 5,296,462, 5,356,816), a cell cycle related gene (WO 01/77354), protein kinase related proteins (WO 01/77356), the LEA-like protein CAP85 (U.S. Pat. No. 5,837,545) and use of a phospholipid binding protein (WO 02/00697). Nevertheless, signal transduction pathways leading to cold acclimation and the identity of the genes that confer resistance to cold stress in plants remain largely unknown.
Yeast has been used for screening plant genes that confer resistance to salt stress. For example, a salt-sensitive yeast strain (JM26) has previously been transformed with a cDNA library from salt-stressed sugar beet and used to screen for clones having increased salt tolerance (WO 02/52012). The transformed yeast cells were grown on a rich medium (YPD) or on a synthetic medium plus methionine and leucine (SD), supplemented with 0.15 M NaCl or with 20 mM LiCl. Putative positive clones showing better growth on the selective media compared to the non-transformed yeast strain were isolated and further characterised. However, the use of yeast for identifying plant genes involved in cold stress has not been used before. A recent study in haploid yeast by de Jesus Ferreira et al. (2001), in which transposon mutagenesis was employed, identified 10 different yeast genes responsive to cold tolerance, which upon mutation caused a growth stop at 15° C. The identified genes include a gene coding for a glutamate synthase (YDL171C), a GTP binding protein (YML121W), a GSK-3 Ser/Thr protein kinase (YNL307C) and a component of TFIID (YLR399C). Three of the genes were previously described as cold responsive (YLR399C, YML121W, YNL307C) and four of the isolated genes were also involved in resistance to salt stress.